Lipopolysaccharide (LPS) constitutes the outer monolayer of the outer membrane of Gram-negative bacteria. As such it forms an important component of the outer membrane and has been considered relevant for vaccine purposes (Verheul et al., 1993). The membrane-anchoring lipid A part is responsible for the well-known endotoxin activity of the molecule (Zähringer et al., 1994).
Such endotoxin activity is undesirable in vaccines. Currently some preparations to be used in vaccines are subjected to rigorous, time consuming and costly purification procedures in order to remove this endotoxin activity prior to their being suitable for use as a vaccine. This allows higher doses due to reduced toxicity. However, drastic purification methods can easily lead to denaturation of protein antigens which need to retain their native conformation in order to induce an appropriate immune response. To date Group A and C polysaccharide vaccines are available which have been rendered substantially free of lipo-polysaccharide by means of purification. To date however no whole cell vaccines substantially free of LPS nor OMP vaccines substantially free of LPS have been produced. The following references provide details of processes used to date in order to avoid LPS in pharmaceutical products Akers (1985), Gabler (1987) and the European Pharmacopoeia, 2nd edition “test for non-pyrogenicity”. Specifically WIIO Tech. Rep. Ser 594:50 1976 deals with the requirements for a meningococcal polysaccharide vaccine.
Mutants with defects in LPS biosynthesis have been described for many bacterial species however none of these have been considered as candidates for a vaccine free of the endotoxic LipidA. All viable mutants retain a minimal lipid A—KDO structure which is the first part to be synthesised (Raetz, 1990) in LPS synthesis. Thus they would not be suitable to overcome the above-mentioned problem facing vaccine producers. Above all, only conditionally lethal mutations have been reported for genes involved in early steps of lipid A biosynthesis in Escherichia coli (Raetz 1990). These mutants have a mutation in genes involved in early steps of lipid A biosynthesis. This finding a suggested that this part of the LPS molecule is in fact essential for bacterial growth. As such this finding would be considered dissuasive by persons skilled in the art of producing vaccines of mutating genes associated herewith as the resulting mutant would not grow. Inhibitors of lipid A biosynthesis have also been found to lead to rapid loss of cell viability in E. coli and several other bacteria (Onishi et al., 1996) thus supporting the above-mentioned hypothesis concerning the essential nature of lipid A biosynthesis.
In addition models for biogenesis of OMPs have been proposed in which their correct folding and targeting is dependent on LPS (Sen and Nikaido, 1991; Reid et al., 1990; Laird et al., 1994; de Cock and Tommassen, 1996).
WO 97/25061 discloses mutants of gram-negative bacteria having a form of LPS deficient in levels of myristic acid moiety, in which the lpxF gene is inhibited.
WO 97/19688 describes mutants of gram-negative bacteria producing less toxic LPS as a result of a mutation in the htrB gene.
We previously cloned the lpxA gene from Neisseria meningitidis which encodes the enzyme UDP-GlcNAc acyltransferase required for the first step in lipid A biosynthesis (Steegh et al. 1997). While attempting to alter the fatty acyl specificity of this enzyme by constructing an E. coli-N. meningitidis hybrid lpxA gene, we made the unexpected discovery forming the basis of the subject invention.